Method and test kit for the separation, purification and recycling of long- and short-chain nucleic acids

ABSTRACT

Long- and/or short-chain nucleic acids are separated, purified and recovered by binding the nucleic acid to a solid phase using a binding buffer, to obtain a bonded nucleic acid, and eluting of the bonded nucleic acid from the solid phase, wherein the binding buffer comprises at least one citric acid salt and at least one alcohol.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to a novel buffer formulation for fast separation,purification and highly efficient recovery of long- and/or short-chainnucleic acids.

2. Discussion of the Background

A multiplicity of commercially-available kits exist today for thepurification and recovery of specific DNA fragments.

All of these methods are based on a method for the preparative andanalytical purification of DNA fragments from agarose gels developed anddescribed for the first time by Vogelstein and Gillespie (Proc. Natl.Acad. Sci. USA, 1979, 76, 615-619). The method combines the break-up (ina saturated solution of a chaotropic salt (NaI)) of the agarosecontaining the bands of the DNA to be isolated with binding of the DNAto glass particles. The DNA fixed to the glass particles is then washedwith a wash solution (20 mM Tris HCl [pH 7.2]; 200 mM NaCl; 2 mM EDTA;50% v/v ethanol) and finally separated from the support particles.

The physico-chemical principle of the systems for isolating nucleicacids on the basis of the binding of nucleic acids to the surfaces ofmineral supports, adopted and commercially available today according tothe background art, should thereby consist in the disruption ofsuperordinate structures of the aqueous medium, through which thenucleic acids on the surface of mineral materials, in particular ofglass or silica particles, adsorb. The disruption of the superordinatestructures of the aqueous medium is thereby always carried out in thepresence of chaotropic ions and is almost quantitative at highconcentrations of these. On this physico-chemical basis described, allcommercially available systems for the isolation of nucleic acidscontain buffer compositions with higher ion strengths of chaotropicsalts, for the binding of nucleic acids to a nucleic acid-binding solidphase.

All the methods described for the isolation of nucleic acids by thebinding of nucleic acids to mineral solid phases by the use ofchaotropic salt solutions have in common the fact that for the bindingof the nucleic acids to the support materials used, high concentrationsmust be adopted. At the same time, chaotropic salts (e.g. guanidinethiocyanate, guanidine hydrochloride, sodium perchlorate or sodiumiodide) are highly toxic potent substances. The buffer systems with veryhigh ion strengths being used often effect a diversion of saltcontaminations, which can prove problematic for a string of downstreamapplications. What is more, in association with chaotropic buffers thereexists a considerable health risk (in particular in long-term use) aswell as a considerable environmental effect through quantities of toxicsubstances dumped into effluent water.

Interestingly, it turns out that all systems which are commerciallyavailable worldwide for isolating nucleic acids on the basis of bindingof nucleic acids to mineral support materials (magnetic particles,membranes, carrier suspensions et al.) work, in principle, according tothe method described. Since the first description by Vogelstein andGillespie, the bound nucleic acids have always been washed with alcoholor salt solutions containing acetone. The wash steps are essentialcomponents of the extraction protocols and, alongside the removal ofbound, undesired, inhibiting substances, always also serve in thenecessary removal of the salts necessary for the binding of the nucleicacids.

In WO 01/62976 A1, a description is disclosed which comprises thepurification of nucleic acids from various reaction assays upon additionof different alcohols, their subsequent precipitation on special solidphases (membranes with specific physical characteristics), wash stepswith alcoholic buffers and the final elution of the nucleic acids bymeans of water.

U.S. Pat. No. 5,405,951 A and EP 0512767 A1 likewise describe theisolation of nucleic acids by incubation of the sample containingnucleic acids with an alcohol, and the subsequent incubation of thesample with a mineral material. The elution of the nucleic acids iscarried out upon the addition of water heated to 60° C.

In DE 10253351 A1, it is disclosed that the purification and recovery ofnucleic acids is carried out by adjusting the solution containingnucleic acid with additives in such a way that it contains monovalentand multivalent cations as well as an alcohol, brings them afterwardsinto contact with the solid phase, subsequently washes the support andreleases the nucleic acid from the solid phase. Ammonium chloride,sodium chloride and/or calcium chloride are used as monovalent saltcomponents, and magnesium chloride, calcium chloride, zinc chlorideand/or manganese chloride are used as multivalent salt components.

It is disclosed that precisely the combination of a monovalent and of amultivalent salt leads to nucleic acids adsorbing on solid phases,wherein the ion strengths necessary for this must only be very small.This has the advantage, if applicable, that wash steps which were alwaysnecessary until now are no longer required, and so the methods forisolating nucleic acids can be clearly shortened and simplified.

At the same time, it emerges that on use of buffer combinations given inDE 10253351 A1 (e.g. magnesium chloride/calcium chloride), thepurification and recovery of DNA fragments from PCR reaction mixturescertainly takes place at a high recovery rate, but unfortunately noselective removal of undesired PCT by-products (e.g. primer-dimers) ispossible.

For this reason, only the general possibility of the recovery of DNAfragments can be demonstrated, but not their efficient purification. Thecause of this observation thereby seems to possibly be the multivalentcation used. If, however, the multivalent cation is removed from thebuffer mixture, then the recovery of nucleic acids with the buffersdescribed is no longer possible on the basis of the very small ionstrengths. However, the use of buffers with only very small ionstrengths was precisely the inventive purpose of the patentspecification.

WO/34463 A1 describes a method for isolating nucleic acids, in which thenucleic acids are bound to a solid phase, wherein the conveyance of thebinding is carried out by binding buffers on the basis of so-calledantichaotropic salts and an alcoholic component. The bound nucleic acidsare washed with wash buffers known in themselves and finally eluted bythe addition of a low-salt buffer. The ion strengths of the so-calledantichaotropic salts come to at least 0.1 M-10M. The so-calledantichaotropic salts for the binding of nucleic acids to a solid phaseare, without exception, chlorides.

WO 89/08257 A1 indicates that citrates belong to the group ofantichaotropic salts. The property characterized in WO 89/08257 A1 is tobe understood in the context of the immobilization of proteins and hasno relationship at all to nucleic acids or methods for the isolation andpurification of nucleic acids.

DETAILED DESCRIPTION OF THE INVENTION

It is an object of the present invention to eliminate the aforementioneddisadvantages of the background art.

This and other objects have been achieved by the present invention asset forth in the claims and specification.

According to the invention, a method and a test kit were produced whichallow a highly efficient recovery of both long- and short-chain nucleicacids, and removing of undesired nucleic acids. The method is simple andfast to carry out.

The invention aims particularly at such uses in which specific nucleicacid fragments from complex reaction assays (PCR, restriction assays,sequencing assays, marking assays) are purified and recovered highlyefficiently and quickly, in order to convey them to a subsequentreaction.

In the context of the present invention, chaotropic salts are salts thatdestroy regular structures of liquid water based on the formation ofhydrogen bonds, in that they inhibit the formation of H₂O cagestructures necessary for salvation. Examples of chaotropic salts arethiocyanates, iodides or perchlorates. They bring about denaturation ofproteins, the increase in the solubility of nonpolar substances in wateras well as the destruction of the hydrophobic interaction.

In the context of the present invention, antichaotropic components aredefined as substances that enhance regular structures of liquid waterbased on the formation of hydrogen bonds. Examples of antichaotropiccomponents are ammonium, sodium or potassium salts. They do not bringabout denaturation, but enhance hydrophobic forces and the increase inhydrophobic interactions.

In the context of the present invention, non-chaotropic components are,for example salts, that are between chaotropic and anti-chaotropicsalts, and include for example, magnesium chloride or aluminiumchloride. Non-chaotropic compounds do not enhance or destroy regularstructures of liquid water based on the formation of hydrogen bonds.Non-chaotropic substances are, for example, those in the middle of theHofmeister series of salts.

In the context of the present invention, alcoholic components are allwater-soluble alcohols such as methanol, ethanol, propanol, isopropanol,ethylene glycol, polyethylene glycol or glycerine. They can be usedalone or in combination.

The method according to the invention for the separation, purificationand recovery of long- and/or short-chain nucleic acids comprises:

-   (1) binding the nucleic acids to a solid phase by using a binding    buffer, to obtain bonded nucleic acids,-   (2) elution of the bonded nucleic acids from the solid phase,    wherein the binding buffer comprises a composition of at least one    citric acid salt and at least one alcohol as effective components.    The binding buffer contains neither chaotropic salts nor a    combination of salts with monovalent and multivalent cations. Salts    with simply positively-charged cations are preferably introduced as    citric acid salts, e.g. corresponding hydrogen citrates or    dihydrogen citrates.

According to the invention, the following salts can be introduced aloneor in combination of at least two salts:

a) Di-ammonium hydrogen citrate,

b) Ammonium dihydrogen citrate,

c) Tri-sodium citrate,

d) Di-sodium hydrogen citrate,

e) Sodium hydrogen citrate,

f) Tri-potassium citrate,

g) Di-potassium hydrogen citrate,

h) Potassium hydrogen citrate.

The alcohol concentrations of the binding buffer are between 20%-90 wt.%, preferably between 40%-70 wt. %. The alcohol concentration includesall values and subvalues therebetween, especially including 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80 and 85 wt. %. Methanol, ethanol,propanol, isopropanol, ethylene glycol, polyethylene glycol or gycerincan be used as alcohols, alone or in combination.

According to the invention, the ion strengths for binding to the solidphase in combination with an alcohol are less than 100 mM, preferablyless than 50 mM. The ion strength includes all values and subvaluestherebetween, especially including 5, 10, 15, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90 and 95 mM.

The solid phase can comprise glass fibre materials, silica gels,suspensions of mineral supports, functionalised magnetic particles;preferably glass fibre materials having a length of from 0.7 μm to 2 μm.

The method according to the invention for the separation, purificationand recovery of long- and/or short-chain nucleic acids includes thefollowing steps:

addition of a binding buffer to a reaction assay containing at least onenucleic acid, to obtain a mixture,

transfer of the mixture from the reaction assay and the binding bufferto a solid phase, and

elution of the nucleic acid bound to the solid phase, and

washing of the nucleic acids bound to the solid phase.

In one embodiment, the binding buffer comprises at least one citric acidsalt and at least one alcohol.

In one embodiment, the nucleic acids bound to the solid phase are notwashed. Even more preferred is a method where no washing is performed atany stage.

Subject of the invention is also a test kit comprising at least onealcohol, at least one citric acid salt, a solid phase and an elutionbuffer.

The use of citric acid salts in combination with at least one alcohol isthe separation, purification and recovery of long- and/or short-chainnucleic acids, in particular of PCR products, restriction assays orsequencing assays.

Surprisingly, the inventor of the present invention has found thatvarious citric acid salts in combination with an alcohol can bind long-and short-chain nucleic acids to support materials, in particular glassfiber materials, and to remove them again.

Surprisingly, the inventor of the present invention has also found thatthe ion strengths necessary for the binding of the nucleic acids mustalso only be present in millimolar concentrations. The use of buffersolutions on the basis of an alcohol and of a citric acid salt allows ahighly efficient recovery of nucleic acids from reaction assays withsimultaneous separation of undesired by-products. This is particularlythe case in the purification of PCR products from PCR reaction mixtures,where primers or primer-dimers in particular have to be separated fromthe specific amplification products.

For example, di-ammonium hydrogen citrate, tri-potassiumcitrate-monohydrate or tri-sodium citrate-dihydrate are introduced aspreferred citric acid salts. Ammonium dihydrogen citrate, di-sodiumhydrogen citrate, sodium dihydrogen citrate, di-potassium hydrogencitrate, potassium dihydrogen citrate can also be introduced. Thecitrates can be used alone or in combination. The ion strengthsnecessary for the binding are smaller than 100 mM, preferably smallerthan 50 mM. The required alcohol concentrations of the binding bufferare between 20%-90 wt. %, preferably between 40%-70 wt. %. Differentalcohols can be introduced; preferably isopropanol is used.

In one embodiment, the method according to the invention for thepurification and recovery of DNA fragments from reaction assays isextremely fast and simple to carry out. The reaction assays from whichthe nucleic acid(s) have to be purified, are mixed with the bindingbuffer according to the invention and subsequently conveyed to acentrifuge column (e.g. with a glass fibre material) and centrifuged.Afterwards, the centrifuge column is brought into a new collectingvessel and the DNA fragments eluted by the column surface after additionof water or a low salt buffer (10 mM Tris HCL).

In contrast to the method (kits) finding commercial use, no wash stepsare necessary. Moreover, the method does entirely without chaotropicsalts which are hazardous to health or the environment, as have beenadopted until now in commercially-available methods.

What is more, the amount of time spent on a purification reaction can bedrastically reduced. A purification can be completed in approx. 3 min,as a rule.

The method makes possible the purification and recovery of a broadspectrum of sizes of DNA fragments with a very high recovery rate.

Surprisingly, the inventor of the present invention has found that thecombination according to the invention of the citric acid saltsinfluences, in terms of ion strength, both the efficiency of therecovery as well as the selectivity regarding the DNA fragments to bepurified regarding their fragment length. This observation can beexcellently used to develop new areas of application. So the methodaccording to the invention also makes possible an efficient purificationof PCR products, restriction assays or sequencing assays. In the case ofsequencing assays, the task is to efficiently separate dye-terminators,with simultaneous efficient recovery of the nucleic acid fragments of awide molecular weight spectrum, in particular also the recovery of verysmall nucleic acid fragments. The implementation of this task-settinglikewise requires only 3 min and is therefore very clearly simpler andfaster than all other methods being used until now.

The difference between the present invention and the solution suggestedin WO/34463 consists in the fact, among others, that in the presentinvention, the ion strengths are smaller than 0.1 M, preferably smallerthan 0.05 M. The use of citric acid salts is not disclosed in WO/34463.This is also explicable as the uses mentioned in WO/34463 cannot at allbe carried out with citric acid salts. The present invention howeverrefers to citric acid salts. WO/34463 describes, without exception,methods for the isolation and purification of genomic nucleic acidswhich are isolated from complex biological samples. But it does notdescribe a method which makes possible the purification and recovery ofa nucleic acid already present. For this reason, the isolation of thenucleic acids is also always carried out with buffers which, alongside asalt component, also contain detergents, proteolytic enzymes as well asfurther additives, the function of which consists in the digestion(lysis) of the biological sample.

The present invention does not refer to the isolation of genomic nucleicacids from complex biological samples. The subject of the presentinvention is the purification of reaction assays, e.g. PCR reactionassays, wherein subsequently e.g. an amplified DNA fragment with a highrecovery rate has to be recovered. No lysis of a biological sample takesplace, in the sense in which this is implemented with the buffers ofWO/34463. WO/34463 describes, without exception, a method for theisolation and purification of genomic nucleic acids, which contains asan obligatory step the washing (several times) of the nucleic acidsbound to the solid phase. Washing is not only necessary in WO/34463 inorder to remove inhibiting substances from the biological samples, it isalso necessary in order to wash out the high salt concentrations of thebinding buffer used. The analysis of the exemplary embodiments showsthat the lysis/binding buffers have ion strengths of >1.5 M, as a rule.The present invention uses clearly smaller salt concentrations (lessthan 0.1 M). For these reasons, the advantage according to the inventionalso consists in getting by without wash steps which have been necessaryuntil now, and thereby clearly simplifying and shortening the operation.

What is more, in the background art there is generally no indicationthat citric acid salts are adopted for binding nucleic acids to mineralsolid phases. The invention is, however, based on precisely thisobservation, all the more so that precisely these salts make it possibleto implement a binding of nucleic acids even in the presence of ionstrengths of less than 100 mM, in particular less than 50 mM. The saltsWO/34463 described do not allow for any binding of nucleic acids andtheir quantitative recovery if they were to be introduced in these lowion strengths.

WO 89/08257 A1 indicates that citrates belong to the group ofantichaotropic salts. The property characterized in this application isto be understood in the context of the immobilization of proteins andhas no relationship at all to nucleic acids or methods for the isolationand purification of nucleic acids.

Other documents alongside the one already described in detail (WO/34463A1) also describe the use of so-called antichaotropic as well aschaotropic salts for the isolation and purification of nucleic acids. Tothis extent it is also not the goal of the present invention to useantichaotropic salts and their application in nucleic acid isolation,but rather much more to create the possibility of using firstly thecombination of citric acid salts and an alcohol in extremely small ionstrengths for an efficient purification and subsequent quantitativerecovery of nucleic acids, and thereby of nucleic acid fragments. Andthis was not known until now.

Having generally described this invention, a further understanding canbe obtained by reference to certain specific examples which are providedherein for purposes of illustration only, and are not intended to belimiting unless otherwise specified.

EXAMPLES

Purification and recovery of a PCR product of 98 bp from a PCR reactionmixture. Use of different binding buffers.

Buffer 1: 50 mM di-ammonium hydrogen citrate/62% isopropanol.Buffer 2: 50 mM tri-sodium citrate-dihydrate/62% isopropanol.Buffer 3: 50 mM tri-calcium citrate-monohydrate/62% isopropanol.Buffer 4: 25 mM di-ammonium hydrogen citrate/62% isopropanol.Buffer 5: 25 mM tri-sodium citrate-dihydrate/62% isopropanol.Buffer 6: 25 mM tri-calcium citrate-monohydrate/62% isopropanol.

In each case, 500 μl of the binding buffer were mixed with 50 μl of aPCR assay with an amplified fragment of 98 bp.

The mixture was subsequently conveyed to a centrifugation column with aglass fibre filter (AF; Fa. Pall), in order to connect the desirednucleic acid fragment, and centrifuged at 10000×g for 1 min. Thecentrifugation column was then inserted into a new 1.5 ml reactionvessel and centrifuged again for 1 min at 8000×g after addition of anelution agent (10 mM Tris-HCL).

The eluted PCR fragments were subsequently analysed on an AgilentBioanalyzer, and the purity as well as the recovery rates weredetermined in relation to the non-purified PCR assays.

No more unspecific primers and primer-dimers could be detected.

The following table contains the recovery rates for each of thedifferent binding buffers P1-P6.

Output fragment; not Binding Binding Binding Binding Binding Bindingpurified buffer P1 buffer P2 buffer P3 buffer P4 buffer P5 buffer P6100% 83.5% 78.5% 76.9% 87.8% 74.4% 82.6%

The example clearly illustrates that very high recovery rates can beachieved with the binding buffers according to the invention.

German patent application DE 10 2005 059 217.1 filed Dec. 7, 2005, andinternational patent application PCT/EP2006/069451, filed Dec. 7, 2006,are incorporated herein by reference.

Numerous modifications and variations on the present invention arepossible in light of the above teachings. It is therefore to beunderstood that within the scope of the appended claims, the inventionmay be practiced otherwise than as specifically described herein.

1. A method for the separation, purification and recovery of a long-and/or short-chain nucleic acid, comprising: binding the nucleic acid toa solid phase by using a binding buffer, to obtain a bonded nucleicacid; and eluting of the bonded nucleic acid from the solid phase;wherein the binding buffer comprises at least one citric acid salt andat least one alcohol.
 2. The method according to claim 1, wherein thebinding buffer does not comprise (i) a chaotropic salt, or (ii) acombination of a salt with a monovalent cation and a salt with amultivalent cation.
 3. The method according to claim 1, wherein thecitric acid salt represents a salt with a singly positively chargedcation.
 4. The method according to claim 1, wherein a hydrogen citrateor a dihydrogen citrate or a mixture thereof is introduced as citricacid salt.
 5. The method according to claim 1, wherein the citric acidsalt is selected from the group consisting of di-ammonium hydrogencitrate, ammonium dihydrogen citrate, tri-sodium citrate, di-sodiumhydrogen citrate, sodium hydrogen citrate, tri-potassium citrate,di-potassium hydrogen citrate, potassium hydrogen citrate and mixturesthereof.
 6. The method according to claim 1, wherein an alcoholconcentration of the binding buffer is between 20%-90 wt. %.
 7. Themethod according to claim 1, wherein an alcohol concentration of thebinding buffer is between 40%-70 wt. %.
 8. The method according to claim1, wherein said alcohol is methanol, ethanol, propanol, isopropanol,ethylene glycol, polyethylene glycol, gycerin or mixtures thereof
 9. Themethod according to claim 1, wherein the ion strength of said bindingbuffer during said binding of said the nucleic acid to said solid phaseis less than 100 mM.
 10. The method according to claim 1, wherein theion strength of said binding buffer during said binding of said thenucleic acid to said solid phase is less than 50 mM.
 11. The methodaccording to claim 1, wherein said solid phase is selected form thegroup consisting of a glass fibre material, a silica gel, a suspensionof a mineral support, a functionalized magnetic particle andcombinations thereof.
 12. The method according to claim 1, wherein saidsolid phase is a glass fibre material having a length of 0.7 μm to 2 μm.13. A method for the separation, purification and recovery of a long-and/or short-chain nucleic acid, comprising: adding a binding buffer toa reaction assay containing a nucleic acid, to obtain a mixture;transferring said mixture to a solid phase, to bind said nucleic acid tosaid solid phase; and eluting said nucleic acid from said solid phase;wherein the binding buffer comprises at least one citric acid salt andat least one alcohol.
 14. The method according to claim 13, which doesnot comprise washing of the nucleic acids bound to the solid phase. 15.The method according to claim 13, wherein the binding buffer does notcomprise (i) a chaotropic salt, or (ii) a combination of a salt with amonovalent cation and a salt with a multivalent cation.
 16. The methodaccording to claim 13, wherein the citric acid salt represents a saltwith a singly positively charged cation.
 17. The method according toclaim 13, wherein a hydrogen citrate or a dihydrogen citrate or amixture thereof is introduced as citric acid salt.
 18. The methodaccording to claim 13, wherein the citric acid salt is selected from thegroup consisting of di-ammonium hydrogen citrate, ammonium dihydrogencitrate, tri-sodium citrate, di-sodium hydrogen citrate, sodium hydrogencitrate, tri-potassium citrate, di-potassium hydrogen citrate, potassiumhydrogen citrate and mixtures thereof.
 19. The method according to claim13, wherein an alcohol concentration of the binding buffer is between20%-90 wt. %.
 20. The method according to claim 13, wherein said alcoholis methanol, ethanol, propanol, isopropanol, ethylene glycol,polyethylene glycol, gycerin or mixtures thereof
 21. The methodaccording to claim 13, wherein the ion strength of said binding bufferduring said binding of said the nucleic acid to said solid phase is lessthan 100 mM.
 22. The method according to claim 13, wherein said solidphase is selected form the group consisting of a glass fibre material, asilica gel, a suspension of a mineral support, a functionalized magneticparticle and combinations thereof.
 23. The method according to claim 13,wherein said solid phase is a glass fibre material having a length of0.7 μm to 2 μm.
 24. A test kit for carrying out the method according toclaims 1 or 13, comprising: at least one alcohol; at least one citricacid salt; a solid phase; and an elution buffer.
 25. A method for thepurification of a PCR product, a restriction assay or a sequencingassay, comprising: contacting the test kit of claim 24 with a PCRproduct, a restriction assay or a sequencing assay.